Nucleic acid synthesis is vital to modern biotechnology. The rapid pace of development in the biotechnology arena has been made possible by the scientific community's ability to artificially synthesise DNA, RNA and proteins.
Artificial DNA synthesis a £1 billion and growing market allows biotechnology and pharmaceutical companies to develop a range of peptide therapeutics, such as insulin for the treatment of diabetes. It allows researchers to characterise cellular proteins to develop new small molecule therapies for the treatment of diseases our aging population faces today, such as heart disease and cancer. It even paves the way forward to creating life, as the Venter Institute demonstrated in 2010 when they placed an artificially synthesised genome into a bacterial cell.
However, current DNA synthesis technology does not meet the demands of the biotechnology industry. While the benefits of DNA synthesis are numerous, an oft-mentioned problem prevents the further growth of the artificial DNA synthesis industry, and thus the biotechnology field. Despite being a mature technology, it is practically impossible to synthesise a DNA strand greater than 200 nucleotides in length, and most DNA synthesis companies only offer up to 120 nucleotides. In comparison, an average protein-coding gene is of the order of 2000-3000 nucleotides, and an average eukaryotic genome numbers in the billions of nucleotides. Thus, all major gene synthesis companies today rely on variations of a ‘synthesise and stitch’ technique, where overlapping 40-60-mer fragments are synthesised and stitched together by PCR (see Young, L. et al. (2004) Nucleic Acid Res. 32, e59). Current methods offered by the gene synthesis industry generally allow up to 3 kb in length for routine production.
The reason DNA cannot be synthesised beyond 120-200 nucleotides at a time is due to the current methodology for generating DNA, which uses synthetic chemistry (i.e., phosphoramidite technology) to couple a nucleotide one at a time to make DNA. As the efficiency of each nucleotide-coupling step is 95.0-99.5% efficient, it is mathematically impossible to synthesise DNA longer than 200 nucleotides in acceptable yields. The Venter Institute illustrated this laborious process by spending 4 years and 20 million USD to synthesise the relatively small genome of a bacterium (see Gibson, D. G. et al. (2010) Science 329, 52-56).
Known methods of DNA sequencing use template-dependent DNA polymerases to add 3′-reversibly terminated nucleotides to a growing double-stranded substrate (see, Bentley, D. R. et al. (2008) Nature 456, 53-59). In the ‘sequencing-by-synthesis’ process, each added nucleotide contains a dye, allowing the user to identify the exact sequence of the template strand. Albeit on double-stranded DNA, this technology is able to produce strands of between 500-1000 bps long. However, this technology is not suitable for de novo nucleic acid synthesis because of the requirement for an existing nucleic acid strand to act as a template.
Various attempts have been made to use a terminal deoxynucleotidyi transferase for controlled de novo single-stranded DNA synthesis (see Ud-Dean, S. M. M. (2009) Syst Synth Boil 2, 67-73, U.S. Pat. Nos. 5,763,594 and 8,808,989). Uncontrolled de novo single-stranded DNA synthesis, as opposed to controlled, takes advantage of TdT's deoxynucleotide triphosphate (dNTP) 3′tailing properties on single-stranded DNA to create, for example, homopolymeric adaptor sequences for next-generation sequencing library preparation (see Roychoudhury R., et al. (1976) Nucleic Acids Res 3, 101-116 and WO 2003/050242). A reversible deoxynucleotide triphosphate termination technology needs to be employed to prevent uncontrolled addition of dNTPs to the 3′-end of a growing DNA strand. The development of a controlled single-stranded DNA synthesis process through Td1 would be invaluable to in situ DNA synthesis for gene assembly or hybridization microarrays as it removes the need for an anhydrous environment and allows the use of various polymers incompatible with organic solvents (see Blanchard, A. P. (1996) Biosens Bioelectron 11, 687-690 and U.S. Pat. No. 7,534,561).
However, TdT has not been shown to efficiently add nucleotide triphosphates containing 3′-O reversibly terminating moieties for building up a nascent single-stranded DNA chain necessary for a de novo synthesis cycle. A 3′-O reversible terminating moiety would prevent a terminal transferase like TdT from catalysing the nucleotide transferase reaction between the 3′-end of a growing DNA strand and the 5′-triphosphate of an incoming nucleotide triphosphate. Data is presented herein which demonstrates that the widely commercially available recombinant TdT sourced from calf thymus is unable to add 3′-O-terminated nucleotide triphosphates in a quantitative fashion (see FIG. 3). In previous reports, the TdT specifically mentioned is recombinant TdT from calf thymus (see Ud-Dean, S. M. M. (2009) Syst Synth Boil 2, 67-73, U.S. Pat. Nos. 5,763,594 and 8,808,989) or uses a different reversible terminating mechanism not located on the 3′ end of the deoxyribose moiety (see U.S. Pat. No. 8,808,989).
Most DNA and RNA polymerases contain highly selective sugar steric gates to tightly discriminate between deoxyribose and ribose nucleotide triphosphate substrates (see Joyce C. M. (1997) Proc Natl Acad Sci 94, 1619-22). The result of this sugar steric gate is the enormous challenge of finding and/or engineering polymerases to accept sugar variants for biotechnology reasons, such as sequencing-by-synthesis (see Metzker M. L. (2010) Nat Rev Genet 11, 31-46 and U.S. Pat. No. 8,460,910). The challenge of finding a polymerase that accepts a 3′-O reversibly terminating nucleotide is so large, various efforts have been made to create reversible terminating nucleotides where the polymerase termination mechanism is located on the nitrogenous base of the terminating nucleotide (see Gardner, A. F. (2012) Nucleic Acids Res 40, 7404-15 and U.S. Pat. No. 8,889,860).
There is therefore a need to identify terminal deoxynucleotidyl transferases that readily incorporate 3′-O reversibly terminated nucleotides and modified said terminal deoxynucleotidyl transferases to incorporate 3′-O reversibly terminated nucleotides in a fashion useful for biotechnology and single-stranded DNA synthesis processes in order to provide an improved method of nucleic acid synthesis that is able to overcome the problems associated with currently available methods.